Martin-Pierre Sauviat

Bistramide A, a new toxin from the urochordata Lissoclinum bistratum Sluiter: Isolation and preliminary characterization

Two cases of human intoxication caused by the lyophilized powder of Lissoclinum bistratum Sluiter, a New Caledonian ascidian, are reported. The symptoms observed were caused by a substance designated bistramide A (C40H68N2O8) of hitherto unknown chemical structure. Preliminary toxicological investigations indicate that bistramide A may effect the central nervous system, leading to paresthesia and loss of muscle tone. A progressive decrease in cardiac rhythm was also observed in animals. Bistramide A (1.4 x 10(-6) M) did not alter the resting potential of frog heart and skeletal muscle but reduced the amplitude and duration of cardiac action potential and prolonged the interval between action potentials. Bistramide A also has a marked cytotoxic effect on cancer cells KB (IC50 = 4.5 x 10(-8) M) and P 388 (IC50 = 2.0 x 10(-8) M) and on normal endothelial cells (IC50 = 2.2 x 10(-8) M). However, it has not been possible to relate the cytotoxic property to the symptoms of intoxication. Bistramide A may originate from the urochordate itself or from symbiotic algae.

Does palytoxin open a sodium-sensitive channel in cardiac muscle?

The effect of palytoxin (PTX) on transmembrane potentials and currents of frog atrial fibres was studied using the double sucrose gap technique. PTX irreversibly depolarized the membrane. This depolarization was reversed only when Na ions were removed from the Ringer solution and replaced by a non-permeant cation such as choline. The depolarization was tetrodotoxin (TTX) insensitive and a function of the external Na concentration. In voltage clamp experiments PTX induced the development of a large inward resting current which did not inactivate and was insensitive to ouabain and to a lowering of the temperature. PTX and ouabain did not share the same receptor. Dose-response curves indicated a stoichiometry of 2, which suggested the aggregation of 2 molecules of PTX to form a channel. The channel formed by PTX remained insensitive to TTX, 4 aminopyridine, tetramethyl-ammonium, Cs and Cd, the classical blockers of Na, K and Ca conductances. PTX reduced the Na current, but not the apparent reversal potential for Na ions. It was concluded that PTX might act on frog atrial fibres as a Na ionophore.

Characterization of the palytoxin‐induced sodium conductance in frog skeletal muscle

1 The effects of palytoxin (PTX) on transmembrane potentials and currents of frog skeletal muscle were analyzed by intracellular microelectrode techniques and the double sucrose‐gap voltage clamp method. 2 PTX irreversibly depolarized the membrane. The depolarization was Na‐sensitive. 3 Under voltage clamp, PTX induced an inward resting current which did not inactivate, was inhibited by external Na+ removal and was a function of external Na concentration. 4 This resting current could be carried either by Na+, Li+, K+ or by guanidinium according to the permeability sequence K+ < Li+ < Na+ < Gua+. 5 The PTX‐induced current was only weakly sensitive to tetrodotoxin. It was reversibly and dose‐dependently inhibited by amiloride with a one to one stoichiometry and a KD of 0.3 mm. 6 Acidic pH partially inhibited the current induced by PTX which was also highly sensitive to external Cd2+ and La3+. The inhibitory sequence for divalent cations was: Mg2+ < Ca2+ = Ba2+ = Mn2+ < Cd2+; with La3+ > Cd2+. 7 The amplitude of the PTX‐induced Irest was markedly reduced in the absence of external Ca2+. 8 PTX induced a Na+ resting conductance in frog skeletal muscle. The size of the channel induced by PTX is larger than the guanidinium ion. External membrane Ca2+ might be a cofactor involved in the mode of action of PTX.

Fumonisin, a toxin from the fungus Fusarium moniliforme sheld, blocks both the calcium current and the mechanical activity in frog atrial muscle.

The effects of fumonisin, a toxin isolated from the fungus Fusarium moniliforme sheld were studied on transmembrane potentials and currents of the frog heart muscle using the double sucrose gap technique. Fumonisin (280 microM) shortened the plateau duration of the action potential. Under voltage clamp conditions, fumonisin inhibited the Ca current. The block occurred without alteration of either the kinetic parameters or the apparent reversal potential of the current suggesting that the toxin blocked the maximal Ca conductance. Fumonisin reduced mainly the phasic component of the peak tension. The time to peak tension was unchanged whereas its relaxation phase was accelerated at positive membrane potentials suggesting that Na-Ca exchange was accelerated by fumonisin. Dose-response curves for the Ca current and the peak tension indicated half inhibition at about 100 microM fumonisin and a stoichiometric parameter of 1.7, suggesting that more than one toxin molecule interacted with the Ca channel. The data suggested that the effect of fumonisin on the Ca current and the tension might account for cardiac failure reported in animal intoxication by Fusarium moniliforme.

Effect of palytoxin on the calcium current and the mechanical activity of frog heart muscle

1 The effect of palytoxin (PTX) on the Ca current (ICa) and the mechanical activity of frog atrial fibres was studied by use of the double sucrose gap voltage clamp technique. 2 In normal Ringer solution, PTX transiently increased the electrically‐evoked peak tension which then decreased while a major contracture developed. PTX slowed the time course of the relaxation phase of the evoked tension. 3 Evidence is presented which suggests that the toxin also increased the entry of Ca and Sr via the Na‐Ca exchange mechanism. It also induced the development of a Ca‐dependent outward current which was inhibited by Sr. 4 In Na‐free solution, PTX increased ICa and shifted the reversal potential for Ca towards more negative membrane potentials, thus suggesting that the internal Ca concentration had increased. Current‐voltage, tension‐voltage, time to peak‐voltage and inactivation time constant‐membrane potential curves were all shifted towards more negative membrane potentials in the presence of PTX. 5 These effects of PTX are similar to those caused by the increase in internal Ca concentration induced by Na ionophores by way of voltage‐dependent Ca influx of the Na‐Ca exchange mechanism.

Effects of ervatamine chlorhydrate on cardiac membrane currents in frog atrial fibres.

1 The effects of a new alkaloid, ervatamine, on transmembrane currents of frog atrial fibres were studied by the double sucrose gap voltage clamp technique. 2 Ervatamine (2.8 × 10−4 m) blocked the action potential without altering the resting membrane potential. 3 The alkaloid depressed the peak INa. The dissociation constant for the blocking effect of ervatamine on gNa fast was 2.35 × 10−5 m with a one to one relationship between the drug molecule and the Na channel. Ervatamine did not alter the apparent equilibrium potential for Na, as well as the activation and inactivation parameters of gNa fast. This suggests that the alkaloid inhibitory effect on gNa can be attributed to a reduction in . 4 Ervatamine prolonged the rate of reactivation of the Na system. It inhibited gNa in a frequencydependent manner; this indicates that the alkaloid acts on open Na channels i.e. that the drug has to enter the channel or cross the membrane to produce the block. 5 Ervatamine inhibited INa slow which occurs in Ca‐free, tetrodotoxin‐containing solutions and moderately decreased Ica which occurs in Na‐free solutions. The drug increased the background K current (IK1) and did not alter the time‐dependent K current (Ix1). 6 The present study shows that ervatamine is a good inhibitor of both fast and slow gNa. This drug also shares some common electrophysiological properties with antiarrhythmic drugs namely: the frequency‐dependent inhibition of the fast gNa and the ability to slow the reactivation of the Na carrying system.

Effect of phoratoxin B, a toxin isolated from mistletoe, on frog skeletal muscle fibres.

The effect of phoratoxin B on transmembrane potentials and currents of frog skeletal muscle was studied using intracellular microelectrode recording and double sucrose gap voltage clamp techniques. Phoratoxin B irreversibly depolarized the membrane. The depolarization was insensitive to tetrodotoxin, tetraethylammonium, 4-amino-pyridine, cesium, cadmium and D-600. In voltage clamp experiments, phoratoxin B induced an inward resting current (Irest) which did not inactivate. Irest was blocked by increasing the external calcium concentration in the Ringer solution; it was not blocked when Sr2+ replaced Ca2+. Analysis of the peak sodium current indicated that while both Ca2+ and Sr2+ screen membrane surface charges, Ca2+ bound and reversed the phoratoxin B induced Irest whereas Sr2+ did not. The inward Irest induced by phoratoxin B developed in the presence of external chloride and remained unchanged in the presence of bicarbonate. The data suggest that the depolarizing action of the toxin might be attributed to an increase in the non-selective leak current and that it might act as a detergent.

Effects of cadmium on the slow inward current of frog heart muscle in relation to a lowering of pH in external solution

1 The effect of cadmium (Cd) on the slow inward current (Isi) of frog atrial fibres was studied by the double sucrose gap technique. 2 Cd (5μm) depressed Isi in a voltage‐dependent manner without alteration of the apparent reversal potential for Isi. 3 Dose‐response curves indicated an apparent dissociation constant for the Cd blocking effect of 4.5 μm at 0 mV, with a one to one relationship between Cd and the slow channel. 4 Increasing the external concentration of Ca ions ([Ca]0) in the tetrodotoxin (TTX)‐containing Ringer solution antagonized the block of Isi by Cd. Double reciprocal plots for Isi versus [Ca]0 drawn in the presence or in the absence of Cd intersected at the ordinate, indicating that Cd competes with Ca for a common binding site. 5 Lowering the external pH from 7.3 to 6.3 depressed Isi. The block caused by H was voltage‐dependent. Double reciprocal plots for Isi versus [Ca]o drawn at pH 7.3 and 6.3 intersected at the abscissa, and indicated that H and Ca did not compete for a common site. 6 Lowering the external pH did not change the ability of Cd to inhibit Isi. 7 The data suggested the existence of two different sites within the slow channel in frog atrial fibres, one of them being H‐sensitive and the other cadmium‐sensitive.

Effect of RP 30356 on the fast inward Na current in frog atrial fibres

The effect of a new drug: RP 30356, on action potential and fast inward Na current of frog atrial fibres was studied using the double sucrose gap voltage clamp technique. The drug (10(-4)M) blocked the action potential without noticeably altering the resting membrane potential. RP 30356 inhibited the fast sodium conductance without changing the selectivity of the Na channel. The time to peak of the inward current was not significantly altered by the drug whereas the rate of the Na current inactivation pi h was slowed. The steady state inactivation membrane potential relationship of the Na system was shifted toward negative membrane potentials by the drug. It was also shown that in Ringer solution the reactivation time constant of the Na system (pi re) was faster at more negative membrane potentials than at more positive ones. The drug increased pi re; the increase was more marked when the membrane was depolarized. The block of Na conductance by the drug was partially removed by increasing the membrane potential to values more negative than -70 mV. The drug did not leave the channel or left it very slowly in the absence of clamp stimulation. The inhibition of the Na conductance by RP 30356 was also frequency-dependent. The data suggest that RP 30356 might be effective in the control of cardiac arrhythmias since it mainly decreased the excitability of depolarized fibres.

Electrophysiological effects of diclofurime on rabbit and frog atrial heart muscle

1 The effects of diclofurime on the electrical activity of the rabbit sinus node, rabbit atria and frog atrial fibres were studied using microelectrode and the double sucrose gap voltage‐clamp techniques respectively. 2 In rabbit sinus node, diclofurime (10−7 m to 10−6 m) decreased the action potential (AP) amplitude and maximum rate of depolarization , increased the AP duration and slowed the sinus rate. 3 In rabbit atria, the drug reduced the amplitude of the depolarizing phase and , lengthened the AP duration and decreased the resting membrane potential. 4 In frog atrial fibres, the drug (10−5 m) depolarized the resting membrane potential, decreased as well as the plateau amplitude. It inhibited the sodium current (INa) with a dissociation constant of 3.7 × 10−6 M and a one to one relationship between the drug molecule and the Na channel. Diclofurime did not alter the apparent reversal potential for the fast Na current (ENa) but it inhibited the sodium conductance (GNa) in a frequency‐dependent manner. 5 Diclofurime also blocked the slow inward current (Islow) without alteration of Eslow. The block of Islow occurred with a dissociation constant of 2 × 10−5 m and unity stoichiometry. 6 The data suggest that diclofurime might be effective in the control of cardiac arrythmias since it exhibited both local anaesthetic‐like and calcium antagonistic properties.